JPS61277631A - Non amide derivatives of somatoclinine and manufacture thereof by gene engineering - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to somatocrinin (5OllatOCrinin).
) and a method for producing the derivative by genetic engineering.

BACKGROUND AND SUMMARY OF THE INVENTION As used herein, "somatocrinin" or rGRFJ refers to both human somatocrinin and animal somatocrinin, and "rGRF (1-43)-X" refers to their non-amide derivatives. here.

X is one amino acid selected from the amino acid group of leucine, glycine, alanine and valine.

Among those derivatives, those in which X is leucine are GRF-O
Also called H.

Human somatocrinin, namely hGR,F, is a peptide composed of a chain structure of 44 amino acids.

The sequence is as follows.

H-T yr -A la -A sp -A la-
, I Ie -P he -T hrG In -G
lu -A rQ -G Iy -A Ia -A r
(1-A Ia-Arg-Leu-NH2, which was recently discovered in extracts of human pancreatic II tumors (
A. Guillemin et al., 5ci
ence, Yudan.

585 = 587.1982).

GRF is particularly active in stimulating the release of growth hormone (GH) both within the tubular mesenteron. Especially in the tube.

Its capacity is several femto moles/1 (ED 50-15 femto moles/i
f). The therapeutic interest of this substance in human medicine therefore lies in the treatment of dwarfism and growth stunting in pediatric medicine. Other applications are also possible in cases of anabolic protein deficiency (stress ulcers, repair of cartilage breaks or lesions, extensive burns - in the anabolic phase - skin repair, bone hyperplasia).

In the field of veterinary medicine, this compound is used for livestock (cows, sheep.

,. ) is of obvious interest in growth and weight gain.

The industrial development of GRF requires the synthesis of significant amounts of it. Conventional solid-phase synthesis methods yield small amounts of this peptide in a short time and at very high cost (Sci
ence, 218. 585-587゜1982)
However, this is incompatible with large-scale pharmaceutical development.

A recombinant DNA having a code for the polypeptide is introduced into a host cell using genetic engineering technology, and the recombinant DNA is expressed (ex.
It is known that it can be manufactured from

However, this direct method is not applicable to the expression of the gene coding for GRF, especially in E. coli (L, sex). This is because GRF is very sensitive to intracellular proteolytic effects.

In the case of somatostatin, this hormone is expressed and protected with a carrier protein, and the somatostatin gene is protected by a single protein located at the end corresponding to the N-terminal part of the gene encoding for somatostatin. It is separated by the ATG codon (code for methionine) from the gene that codes for the carrier protein ((takura et al., 5ci).
ence 1977, Yucho.

1056-1063). After expression, the hybrid protein can be cleaved at the methionine level using cyanogen bromide to release somatostatin.

Cyanogen bromide also stimulates cleavage of the desired product molecule, so this method is not applicable to GRF.

Although cleavage of polypeptides at other amino acid levels has been described, such methods do not have the specificity necessary to obtain products of sufficient purity in satisfactory yields.

GRF (1-43)- where X is defined above
The XI derivative was linked to a carrier protein via a tryptophan residue. It has now been found that the methods of genetic engineering that allow the synthesis of such derivatives allow them to be produced on an industrial scale. The resulting hybrid protein is then cleaved at the tryptophan level to provide the M conductor.

Such cleavage occurs selectively at the tryptophan level by the action of oxidizing agents such as 2-(2-ditrophenylsulfenyl)-3'-promo-3-methylindolenine, hereinafter BNPS-skatole. It is done.

From one point of view, the present invention is a method for obtaining GRF-OH by genetic engineering.

1) Trp-GRF (1-43>-Xk: step of constructing a DNA fragment having a pair of surcodes.

2) A step of inserting the fragment into a vector containing the gene encoding the carrier protein, together with genetic elements necessary for its expression, and this insertion involves the insertion of the gene encoding the terminal carboxyl portion of the carrier protein. What you do at the level of an array with .

3) Transforming host cells with the generated recombinant vector.

4) Expressing the polypeptide of Formula I.

In the formula, X=Leu, Ala, Vat or Gly
PrOt=residues of the protein 5) Recover the polypeptide of formula l and cleave it at the tryptophan level using an oxidizing agent.

The formula GRF (1-43)- where X is defined above
Step of obtaining compound X.

The present invention relates to a method including the steps of.

Trp-GRF (1-43
) - The DNA fragment that carries the code for X is.

- chemically synthesizing the oligonucleotide carrying the tryptophan codon.

- GRF carried by the vector (
1-43) Step of connecting to the gene having the code for -X at the end corresponding to the N-terminal part.

- and a step of isolating the DNA fragment obtained above by a conventional method.

It is obtained by a method including each step of.

The synthesized oligonucleotide was Trp-GRF (
1-43> The reading phase of the gene encoding for -X is selected to fuse with that of the carrier protein.

The gene that codes for GRF (1-43) -X, ie GRF-OH, where X is leucine.

Preferably, the commercially available plasmid pUc9 (Messin
g and Vielra, Gene, 1
9° (1982) 259). It contains the gene encoding the 9th order methionine-○RF-01-1.

0M3-8-CI-12-CH,, -CH-C0-()
RT'-OH(II)■H2 The second step of the method of the present invention is to convert the obtained DNA fragment into
This involves inserting the gene encoding the carrier protein into a vector containing the gene encoding the carrier protein along with the genetic elements necessary for its expression. A carrier protein is a protein that is efficiently expressed by host cells. This carrier protein is preferably β-galactosidase when the host cell is E. coli.

In one way, Trp-GRF (1-43)
The DNA fragment encoding for -X is inserted into a vector containing the gene for the carrier protein and the genetic elements necessary for expression.

Alternatively, the fragment of DNA is inserted into a vector containing the gene encoding the carrier protein and the resulting gene isolated.

inserted into a new vector containing the genetic elements necessary for expression.

The host mll1 used in the method of the invention is a prokaryotic or eukaryotic cell. The preferred prokaryotes are the bacterial species Escherichia coli (
5cherichia G of i).

An example of a suitable cell is CAG1139 (Gross
man A, D, et at, , Ce1l
, 32 (1983), 151-159) and M C1061 (Casadaban M, et
al., J., Bacteriol.

(1980), Eng., 971-980).

From another point of view, the present invention relates to GRF (1-43
)-XkJ:Structure 6. This invention relates to a recombinant vector containing a code for a high-7 hybrid protein.

A preferred recombinant vector binds GRF (1-43)-X at the terminal carboxyl side via tryptophan. Contains a DNA base sequence having a code for β-galactosidase, an analog thereof, or a fragment thereof.

Regarding expression (transcription and translation), a recombinant vector is a vector that contains suitable genetic elements by conventional genetic engineering techniques. placed under l. Gene expression is condition dependent, i.e. chemical inducers2 e.g. IP
by TG (isopropyl-thio β-D-galactoside) or by physical conditions.

For example, activation by temperature is desirable.

Then, the regulation element
The gene is introduced into a DNA vector along with ent),
Transformed host cells are obtained and selected.

°The DNA vector is preferably a plasmid.

The transformed aWa is isolated by conventional techniques.

It is cultured under conditions that allow expression of the genes to obtain the desired hybrid protein.

The present invention also includes: It relates to a strain of host cell containing a recombinant vector as defined above. Examples of such strains include preferred recombinant vectors (plasmid
202.17) The strains of E. coli M C1061 containing the following are listed below. It was deposited with the National Microbiological depository in Paris as No. 1-354 on October 22, 1984 (dep
posed at the ” Co11e
ctionNatiOnale de CUltU
reS de Micro-organisme
s” in Paris, on 0cto
rbe 22゜1984 tlnder
No, i-354n.

In another aspect, the present invention relates to a hybrid protein of the following formula.

NH2Prot CONHCHCOGRF (143)
X where X is one of the amino acids mentioned above.

prot represents the residue on the terminal carboxylic acid side of the carrier protein.

A preferred hybrid protein is a compound of formula I above, with Prot being β-galactosidase.

It is the terminal carboxylic acid residue of an analog or fragment of this molecule.

The hybrid protein is extracted from the culture medium or transformed cells and purified, eg, by chromatography.

The hybrid protein of formula 1 obtained in this way is 2
cleaved with BNPS-skatole (H
, M., 5teiniann, J., Biol.

Chem, 1978. '251.8708-87
20>, derivative GRF (1-43) -XG[chi+
7) Method ICJ:)) Extraction.

N was prepared by gel filtration and HPLC (R.

Quillemin et at, , 5cie
nce 1982. Yudan.

185-187).

In another aspect, the present invention relates to polypeptides of the following formula:

GRF (1-43) -X (I I) where X is one amino acid chosen from leucine, valine, alanine or glycine, which is the key intermediate to obtain GRF.

The following examples are illustrative of the invention without limiting it. The experimental details of genetic engineering techniques are described in the literature (Molecular CIoning
by T. Maniatis, Ed.
, Co1d Spring 1arbor
Labs, USA, 1982).

Double helix oligonucleotides have a base sequence of 5'-0
The first oligonucleotide, which is phosphorylated at the 5' end with H-ACCACAGCTGG, is
-0H-AATTCCAGCTGTGGT by hybridization with a second oligonucleotide.

This oligonucleotide is then treated with the restriction enzyme Rsa
I opened the front with l. Plasmid containing the gene encoding Met-GRF-OH 1) UO3
double helix DNA (CREATIVE B l0
M0LECULES, San Francisco,
Ca, 94080, USA) (Figure 1)
to fuse with. The oligonucleotide is GRF-OH
at the level of the second nucleotide of the gene that codes for and its complementary nucleotide (Figure 2)
. The fusion is carried out in the presence of the DNA ligase of Batatteriopha-di T4.
This is done by culturing the enzyme-opened plasmid pUc9 with a 50-fold molar excess (with respect to the l end) and the enzyme-opened plasmid pUc9.

The new double helix DNA obtained in this way is...

Contains the gene encoding Trp-GRF-OH. After purification by gel electrophoresis, the restriction enzyme 5al
Due to the action of l, the D shown in Figure 2 containing this gene exactly
NA fragments are obtained. This fragment is then cloned into E. coli (strain MC1061) using the EcoRI-8all large fragment of plasmid ρ8R322 as a vector.

Plasmid pDIA 5501 (P, Le
Platoisand A, Danchin
Biochimie, 1983.65°317-, 3
24) is a plasmid derived from plasmid I)BR322 and contains the gene encoding β-galactosidase. This plasmid pDIA
5501 was cleaved with restriction enzyme S ac I.

Then, 5aCI from plasmid pM C1396
I-l: By fusion with coRIli fragment, the gene is modified by placing EOORI side (M, Ca5a
daban et al., J., 3acteri.
o1. (1980) Yugumi: 971-980). Modified plasmid pDIA5501.

Contains the gene encoding β-galactosidase truncated from the C-terminus. This again.

origin of replication and pB R322 ampicillin (a
mpicilline) contains genes that code for resistance.

11. The plasmid obtained in step a is digested with restriction enzymes EcoRI and 3all, and the DNA fragment containing the gene encoding Trp-hGRF-OH is purified by gel electrophoresis.

This fragment was then converted into bacteriophage T4 DNA.
Modified plasmid pD I A 5s in the presence of ligase
ol and was previously digested entirely with enzyme 5all and partially with enzyme EC0RI.

It is cultured with the EcoRI-8alI fragment.

A new plasmid, plasmid 202.17, is obtained.

Plasmid 202.17 contains the DNA fragment shown in FIG. 2 and has the outline shown in FIG.

- EC0RI of modified plasmid pDIA 5501
- elements contributed by the 3all fragment.

-・pB R322 replication origin.

・5alI-E from plasmid pB R322, which carries the gene encoding ampicillin resistance
coRI fragment.

- Gene encoding the heat-sensitive repressor Cl857.

- Promoter PR of bacteriophage λ.

- Between the nucleotide encoding the N-terminus of the protein and the cleavage site of the enzyme Sacl.

EcoRl-3acl fragment carrying the part of the gene containing the gene encoding β-galactosidase.

- Plasmid I) MC1396-derived 5acl -
A gene encoding for Tr-GRF-OH, an element provided by the EcoRI-8all fragment of the plasmid obtained with ECORICOR-1,a.

Includes each element of

Plasmid 202.17 is then Trp-GRF-
It carries the gene encoding a hybrid protein consisting of a modified β-galactosidase linked to OH, and its expression is dependent on the promoter PR, which itself is under the control of repressor C857.

strain containing plasmid 202.17.

Cultured in 15 liters of medium containing 50 μg/■1 ampicillin at 30° C. until a density corresponding to io, ooo, ooo bacteria/1 was obtained, and then.

Incubation is continued for 2-3 hours at 42°C. Hybrid protein synthesis was performed by electrophoresing cellular proteins on sodium dodecyl sulfate (SDS)-polyacrylamide gels, followed by Coomassie blue (Coomassie blue) gel electrophoresis.
When stained with blue) and analyzed.

This is evidenced by the appearance of a specific band corresponding to the size of β-galactosidase.

Centrifuge 20 ml of bacterial culture. Take up the button in 3 ml of 10011M phosphate buffer at p) 17.8.

The bacteria thus brought back into suspension are sonicated.

The resulting bacterial lysate was centrifuged.

A first supernatant Sl and a button C1 are obtained. button C1 to 0
．． 1% alkylaryl
Taken in an ester type (Triton) detergent 11,
The resulting suspension is centrifuged to obtain a second supernatant S2 and button C2. Take button C2 in 0.5 ml of 1% 5DS and centrifuge the resulting suspension to obtain a third supernatant S3 and button C3. Take button C3 in 80.5 ml of 1% 5D, centrifuge the resulting suspension and remove the fourth supernatant $4.
get.

Combine supernatant 82, 83 and S4. This pool (Pool A) is gel filtered on a Sephadex G25 gel adjusted with 70% acetic acid. Collect the elution fractions,
Continuous measurements of optical density (OD) are made at 280 nm.

The fractions eluted in the dead volume of the column and the fractions corresponding to the protein peak are combined. This pool (Pool B) contains the hybrid protein.

The protein concentration of pool B was determined using the Bradford method (B
1O-RAD Protein As5ay Kit
). To pool B, BNPS-skatole (PIERCEC
chemical , Go , , Rockford
, I l, USA) and keep it away from light.
While stirring intermittently.

After 24 hours of contact at room temperature, pool B is extracted five more times with diethyl ether. The final aqueous phase obtained from this extraction is subjected to column chromatography (PERKIN-
ELMER/I- (S-5C18 column). 2 types II
Solution I (buffer A: 99.9% H20 and 0.1% trifluoroacetic acid (TFA): !l[[B: 9
9.9%7t Tonito IJ/L, O and 0.1%TFA
) at a flow rate of 21/min for 15 minutes using a linear gradient (buffer 8
15% to 80% buffer).

Collect the elution fractions and analyze at 220 nm (specific for peptide binding)
``Continuously perform C'OD measurements.

The elution curve of the obtained fractions is shown in FIG. Here, the x-ring is the elution time in minutes and the y-axis is the optical density OD. Shift 1
i1A represents gradient elution as defined above.

1, f Identification of hGRF-0) 1 The elution fractions collected in a certain bottle were freeze-dried (TF
(For removal of A) L, taken in 50 μm distilled water and RI
A (radioimmunoassay) type radioimmunoassay was used.

This RIA test was carried out in the presence of small amounts of antibodies produced against hGRF (1-40) and hGRF-OH obtained according to the present invention and hGRF (1-40) obtained by chemical synthesis and labeled with 1251.
It is based on competition with GRF-0)-1.

As shown in FIG. 4, it was confirmed that hGRF-OH obtained according to the present invention was contained in the fractions collected at the elution time between 3 minutes and 7 minutes and 30 seconds.

This figure shows that two fractions of GRF-OH components are collected at elution times between 3 and 8 minutes.

The y-axis on the right side of the figure has a corresponding scale (GRF-OH determined by RIA, 1107ml) t''.

1.0L gloss 11 Biological activity tests were performed on one of the above fractions.

In this study, each of three rats was given h
0.5 μQ of GRF-OH was given intravenously. 0.5 of hGRF obtained by chemical synthesis in each of three other rats.
μg was given intravenously.

At +2, +5, +10, +15, and +30 minutes (from the time of injection), samples of growth hormone contained in the plasma were prepared for each rat (by intracardiac sampling). The activity curve obtained from this measurement shows that hGRF-OH obtained according to the present invention was effective in stimulating hGRF-OH in rats.
It is shown to cause a reaction similar to that induced by RF.

[Brief explanation of drawings]

Figure 1 shows the DNA base sequence of plasmid pUc9. Figure 2 shows a DNA fragment containing the gene for Trp-G RH-OH, and Figure 3 shows an outline of plasmid 202.17. Figure 4 shows the results of chromatography and RIA:twA.

Claims (11)

[Claims] (1) A method for obtaining by genetic engineering a non-amide derivative of somatocrinin of the formula GRF(1-43)-X, where X is leucine, glycine, valine or alanine, wherein 1, X is as defined above. , Trp-GRF(1
-43) A step of producing a DNA fragment having a code for -X, 2. A step of inserting the fragment into a vector containing a gene having a code for a carrier protein together with genetic elements necessary for its expression, This insertion is carried out with a sequence encoding the terminal carboxyl portion of the carrier protein. 3. Step of transforming host cells with the resulting recombinant vector. 4. Process of expressing polypeptide of formula I, ▲There are mathematical formulas, chemical formulas, tables, etc.▼(I) In the formula, X=Leu, Ala, Val or GlyPro
t = residue 5 of the carrier protein, the polypeptide of formula I is recovered and it is cleaved at the tryptophan level using an oxidizing agent to obtain the formula GRF(1-43) where X is as defined above. -Process of obtaining the compound of X. A method that includes each step of. (2) A DNA fragment having a code for Trp-GRF(1-43)-X is obtained by: 1) chemically synthesizing an oligonucleotide carrying a tryptophan codon; 2) converting the oligonucleotide to GRF (
1-43) - In the gene that has the code for
2. A method according to claim 1, wherein the method is obtained by ligating the distal portions at corresponding ends. (3) The method according to claim 1 or 2, wherein the carrier protein is β-galactosidase, an analog thereof, or a fragment thereof, and the host cell is E. coli. (4) The hybrid protein is Prot and
A code for a hybrid protein consisting of GRF-OH, which corresponds to the following formula as defined in claim 1 and is bound to a carrier protein via tryptophan at its N-terminus: A recombinant vector with ▲There are mathematical formulas, chemical formulas, tables, etc.▼(I) (5) The recombinant vector according to claim 4, wherein the carrier protein is β-galactosidase. (6) The recombinant vector according to claim 5, which is plasmid 202.17. (7) A hybrid protein of the following formula, wherein X is as defined in claim 1, and Prot is a residue of the carrier protein. ▲There are mathematical formulas, chemical formulas, tables, etc.▼(I) (8) Prot is a residue of β-galactosidase,
A hybrid protein according to claim 7. (9) A polypeptide of formula GRF(1-43)-X, wherein X is leucine, alanine, valine or glycine. (10) A strain of E. coli containing the recombinant vector according to any one of claims 4 to 6. (11) The strain according to claim 10, which was deposited with the National Microbial Depositary in Paris as No. I-354 on October 22, 1984.